Neuropathic pain is prevalent, persistent, and debilitating. There is no effective treatment without untoward side effects for the neuropathic pain syndromes that afflict an estimated 5 million patients in the U.S.including patients with diabetes, HIV,the elderly, and young people who experience trauma. In this revised competitive renewal proposal, we apply data from the previous funding period to investigate the following hypothesis: Peripheral nerve injury activates central innate immunity via microglial Toll-like receptor 4(TLR4) and CD14 expression that leads to astrocytic activation and chemokine expression which in turn manifests as persistent neuropathic pain. Microglia play a major role in initiating the cascade while astrocytes provide the glia-to-neuron signal to maintain pain states. Three important recent findings from our work direct this current proposal: 1) Intense co-stimulatory molecule B7.2 expression but not B7.1 was observed in the lumbar spinal cord following nerve injury. These data suggest a role of protective CMS autoimmunity following peripheral nerve injury. 2) Evidence supporting the role of innate immunity in neuropathic pain, i.e. involvement of TLR4. 3) Data to demonstrate a key role of one chemokine, monocyte chemoattractact protein (MCP)-1 in nerve injury-induced allodynia. The central hypothesis will be tested by using established methods in our laboratory to investigate the following Specific Aims: 1) Assess the role of accessory molecules CD14 and MD-2 in the microglial TLR4 responses after nerve injury. 2) Determine whether TLR4 and CD14 result in spinal chemokine expression. 3) Determine the role of the glial-derived chemokine, MCP-1 as a critical regulator of leukocyte trafficking following nerve injury that results in behavioral hypersensitivity. Genetically altered mice, antisense ODN, neutralizing antibodies, immunohistochemistry, RT-PCR, Western Blot analysis, FACs and nociceptive behavioral assays will be used to resolve these specific aims. The results from these multidisciplinary studies may culminate in novel pharmacopeia to treat and prevent chronic neuropathic pain and thus, has the potential for high clinical impact.